This and several other laboratories have shown that nonglycosylated and glycosylated proteins in cells infected with membrane maturing viruses assemble into plasma membranes by different pathways. We have also shown this is true of the glycosylated and nonglycosylated plasma membrane proteins of uninfected HeLa cells. In one review (Rothman and Lenard, 1977) these proteins have been termed "endo" and "ecto" proteins postulating an inside (the plasma membrane) and outside disposition respectively for proteins assembled by different pathways. Some nonglycosylated proteins, of which the matrix protein of vesicular stomatitis virus is an example appear to be inserted directly from soluble pools after synthesis. The glycosylated proteins by contrast require an internal membranous carrier. We will continue our efforts to isolate an internal membranous component which is carrying glycoprotein to the plasma membrane in order to assess its composition. We propose to study which nonglycosylated proteins in uninfected cells are rapidly synthesized and inserted into plasma membranes. Thus we will study the synthesis and association of the two major rapidly synthesized nonglycosylated proteins of HeLa plasma membranes of molecular weights approximately 43,000 and approximately 34,000 and are approximately 7% and 9% of the total membrane protein respectively. We have shown that in addition to the well known sialic terminating heterosaccharides that these is a second major class of oligosaccharides N-glycosidically linked to membrane glycoproteins. Rapidly growing and transformed cells express a greater proportion of the second category--an array of neutral mannose yields N-acetylglucosamine containing glycopeptides whose size of the oligomannosyl cores is also growth dependent. We will purify in chemical amounts single species of such neutral glycopeptides from purified glycoproteins isolated from rapidly growing density inhibited and transformed cells and performed structural studies using gas liquid chromatography, methylation analysis, periodate degradation. Purified Sindbis virus glycoproteins and various endo-beta-N-acetylglucosaminidases ("endo-H", "endo-D", "Endo-C II") will be used to aid these studies.